: Heptitis C virus (HCV) is the most common cause of chronic viral hepatitis in the United States. In the infected host, both CD4 and CD8 T cell responses directed at HCV are thought to be important in both the host's control of viral replication and the liver pathology that results from the ongoing T cell attack against virus persisting in the liver. Despite the central role of T cells in the immune pathogenesis of chronic hepatitis, technical limitations have so far prevented the acquisition of definitive data on the most basic features of the anti-HCV T cell response in infected individuals, the frequency of the specific T cells in vivo, their fine specificity (the actual peptide-determinants they recognize), and their effector class (defined by their cytokine expression patterns and cytolytic activity). Without the ability to define clearly these basic parameters of T cell-mediated immunity, our understanding of protective vs. pathogenic T cell response types in HCV infection remains incomplete, and this precludes studies aimed at altering the antiviral immune response and preventing or treating HCV-related disease. It is hypothesized that the complete clonal size (the magnitude) and fine specificity of the HCV-core-antigen-specific T cell response can be assessed, irrespective of the patient's HLA haplotype, by ex vivo ELISPOT assays that test an overlapping 10-mer peptide series that walks the core protein sequence in steps of single amino acids. (The core protein was selected to demonstrate the feasibility of this approach). In Aims 1 and 2, therefore, it is proposed to perform systematic ELISPOT determinant mapping with an overlapping HCV-core peptide series. It is further hypothesized that measurements of cytokine coexpression patterns in two-color ELISPOT assays are suitable for characterizing the quality of the core-antigen-specific CD4 and CD8 T cell response and that cytokine coexpression in the specific cells studied at single-cell resolution proves to be more highly regulated than predicted by the Th1/Th2, Tcl/Tc2 model. To test this hypothesis, in Aim 3, it is proposed to measure the coexpression of cytokine and granzyme B in individual, freshly isolated HCV-peptide-reactive memory cells. Finally, by defining repeatedly, over the 5 years of this project, in individual HCV-infected patients the magnitude, the fine specificity and the cytokine quality of the HCV-core-protein-specific T cell response. In Aim 4, the stability of the T cell response will be tested, thus addressing the hypothesis that the T cell repertoire directed at this HCV antigen can be dynamic, shifting the determinants targeted.